CN116479043A - Extracellular vesicle co-delivery system for tumor treatment and application thereof - Google Patents
Extracellular vesicle co-delivery system for tumor treatment and application thereof Download PDFInfo
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- CN116479043A CN116479043A CN202211546236.6A CN202211546236A CN116479043A CN 116479043 A CN116479043 A CN 116479043A CN 202211546236 A CN202211546236 A CN 202211546236A CN 116479043 A CN116479043 A CN 116479043A
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- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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Abstract
The invention belongs to the technical field of anti-tumor drug research, and particularly relates to an extracellular vesicle co-delivery system for tumor treatment and application thereof. The co-delivery system is obtained by encapsulating effector molecule mRNA related to the mode of cell death in an extracellular vesicle membrane, incorporating a substance capable of inducing the generation of singlet oxygen into the extracellular vesicle membrane, and loading a targeting peptide or antibody on the surface of the extracellular vesicle. The extracellular vesicle co-delivery system provided by the invention has the advantages that effector molecule mRNA is inhibited by optimized puromycin translation in an extracellular vesicle donor cell, and mRNA encapsulation to the extracellular vesicle is promoted; in donor cells, effector proteins are efficiently expressed, inducing tumor cell death, with dilution of puromycin concentration and inactivation of puromycin by treatment with a substance capable of generating singlet oxygen.
Description
Technical Field
The invention belongs to the technical field of anti-tumor drug research, and particularly discloses an extracellular vesicle co-delivery system for tumor treatment and application thereof.
Background
Tumors are important diseases affecting human health and have become the second leading cause of death worldwide. Tumor treatment mainly comprises traditional operation treatment, radiation treatment, chemotherapy and novel treatment modes such as targeting treatment, immunotherapy and the like which are rapidly developed in recent years. Targeted drugs have been hot spots in the development of antitumor drugs, and can be broadly divided into two classes, monoclonal antibodies and small molecule compounds. In recent years, with the deep research, new targets are continuously emerging, and anti-tumor drug development is greatly advanced. At present, the U.S. food and drug administration has approved tumor targeted therapeutic drugs for more than 30 targets, many of which are subjected to batch treatment for multiple indications, and the appearance of the drugs brings new hopes for tumor patients, but the drugs still have the problems of large therapeutic dose, high administration frequency and the like, and bring inconvenience to further transformation.
Extracellular Vesicles (EVs) are natural nanoparticle biological carriers with many advantages in terms of biocompatibility, immunogenicity, stability, pharmacokinetics, biodistribution and cellular uptake mechanisms. Because extracellular vesicles "recognize" specific cells, extracellular vesicles deliver therapeutic drugs with better efficacy and less off-target effect than other biological carriers (e.g., liposomes). In recent years, more and more studies have shown that extracellular vesicles have great potential in tumor therapy. The mRNA vaccine is one of nucleic acid medicines, and the nucleic acid medicines treat patients from the gene level, and the corresponding medicines can be developed only by developing proper sequences aiming at target genes, so that blindness in the development process is avoided. The nucleic acid molecules are degraded in the body fluid circulatory system, have immunogenicity and are easily enriched in organs such as liver. Extracellular vesicles have many advantages as novel nanomedicine delivery systems, and extracellular vesicle-binding nucleic acid therapies have become a new hotspot. The effector molecules of cell death modes such as apoptosis, cell coke death, iron death and the like can directly kill tumor cells, thereby achieving the treatment effect. Thus, starting from effector mRNA in a cell death mode, an engineered extracellular vesicle targeted for delivery of mRNA drugs is at the forefront for effective treatment of tumors and reduction of side effects.
Disclosure of Invention
In order to solve the technical problems, the invention provides an extracellular vesicle co-delivery system for tumor treatment, which can effectively treat tumors and has the advantages of small side effect, high stability and high killing efficiency.
The first object of the present invention is to provide an extracellular vesicle co-delivery system for tumor therapy, which is prepared by the following steps:
s1, transfecting cells with a recombinant expression vector of a tumor targeting protein or targeting peptide to obtain cells transfected with the recombinant expression vector of the tumor targeting protein or targeting peptide;
s2, transfecting a recombinant expression vector containing a cell death related effector molecule plasmid into cells transfected with tumor targeting proteins or targeting peptides in S1, adding purinin to inhibit translation of the transfected effector molecule plasmid, and centrifuging to obtain extracellular vesicles with surface modified effector molecule mRNA;
and S3, incubating the extracellular vesicles obtained in the step S2 with a substance capable of generating active oxygen to obtain an extracellular vesicle co-delivery system.
Preferably, the tumor targeting protein or targeting peptide is a HER2 single chain antibody or a GE11 peptide.
The effector molecule related to cell death is one of tbid, GSDMD-N, GSDME-N or GSDMB-N.
Preferably, the recombinant expression vector of the tumor targeting protein or targeting peptide is obtained by loading the tumor targeting protein or targeting peptide between BamHI and HindIII cleavage sites of a recombinant plasmid pcDNA3.1-Lamp2b containing Lamp2b genes;
the recombinant expression vector containing the cell death related effector molecule plasmid is obtained by loading the effector molecule plasmid between BamHI and HindIII cleavage sites of a pcDNA3.1 (+) vector.
Preferably, the transfection in S1 is to mix the recombinant expression vector of the tumor targeting protein or targeting peptide with the transfection reagent according to the ratio of 1-2 mug to 2-4 mug, and then to add the mixture into a cell culture system without double antibody culture medium for culturing for 24 hours after standing for 20-40 min at 37 ℃;
the transfection in S2 is to mix the recombinant expression vector containing the cell death related effector molecule plasmid with a transfection reagent according to the ratio of 1-2 mug to 2-4 mug, and then to add the mixture into a cell culture system of the recombinant expression vector transfected with tumor targeting protein or targeting peptide in S1 after standing for 20-40 min at 37 ℃.
Preferably, the specific procedures for inhibiting effector plasmid translation following transfection are:
after adding the purine, incubating for 4-6 hours at 37 ℃, and continuously incubating for 48 hours in a complete medium without serum, and collecting cell supernatant;
the centrifugation is to use 100000 Xg ultracentrifugation for 10-16 h on the cell supernatant after removing cell debris.
Preferably, the inhibitory concentration of the purlin is 4 μg/mL.
Preferably, the substance capable of generating active oxygen is a sonosensitizer or photosensitizer.
Preferably, the sonosensitizer is chlorin E6 or purpin 18.
Preferably, the co-incubation is carried out by mixing the extracellular vesicles obtained by the S2 and substances capable of generating active oxygen according to the mass ratio of 1:10-50, and then co-incubating for 12-24 h.
The second object of the invention is to provide an application of the extracellular vesicle co-delivery system in preparing an anti-tumor drug.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention modifies the tumor targeting protein or targeting peptide on the surface of the extracellular vesicle, mixes the tumor targeting protein or targeting peptide with the transfected recombinant expression vector containing effector molecule plasmid, then utilizes puromycin with proper concentration to inhibit the translation of the transfected effector molecule plasmid, and harvest the extracellular vesicle with more effector molecule mRNA; and substances (such as sound sensitizers, photosensitizers and the like) capable of generating active oxygen are utilized, so that puromycin is inactivated in the receptor cells, and effector molecule mRNA is normally translated in the receptor cells, and the killing effect is actively exerted. The extracellular vesicle co-delivery system provided by the invention can effectively treat tumors, and has the advantages of small side effect, high stability and high killing efficiency.
2. The targeting protein or the targeting peptide used in the invention can recognize the relevant antigen of diseases, and effector molecule mRNA kills tumor cells; the addition of puromycin to donor cells inhibits the expression of effector mRNA in donor cells; the addition of a substance capable of generating active oxygen to extracellular vesicles can inactivate puromycin in the recipient cells, so that effector mRNA is normally expressed in the recipient cells, thereby killing tumor cells.
Drawings
FIG. 1 is a screen for puromycin concentration;
FIG. 2 shows the Western blot detection of extracellular vesicle P1h3 expression;
FIG. 3 is the effect of each group on tumors in SKBR3 tumor-bearing transplanted mice.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will now be further illustrated with reference to specific examples, which are given solely for the purpose of illustration and are not to be construed as limiting the invention. The test specimens and test procedures used in the following examples include those generally under conventional conditions, or under conditions recommended by the reagent company, if the specific conditions of the test are not specified in the examples; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
Example 1
Acquisition of extracellular vesicle co-delivery System targeting HER 2-positive tumors with the apoptosis-related effector molecule GSDMD-N mRNA
The specific operation process is as follows:
s1, constructing a recombinant expression vector pcDNA3.1-P1h3-Lamp2b: the recombinant expression vector is completed by Nanjing Jinsri biotechnology company;
(1) Obtaining the P1h3 Gene sequence
Designing a primer pair, and amplifying the obtained product by polymerase chain reaction to obtain P1h3, wherein the sequence of the upstream primer added with BamH I enzyme cutting site is F-BamH I:5'-CTCGGATCCGCGCCACCATGACCTGCATGTTGGAACGCAT-3', as set forth in SEQ ID NO:1 is shown in the specification; the sequence after the downstream primer is added with HindIII cleavage site is R-HindIII: 5'-CTCAAGCTTGGCTCACTTGTCGTCATCGTCTTTGTAGTCCCCCTCACTCCT CGCAGCACAGT-3', as set forth in SEQ ID NO: 2.
Wherein, the sequence of the amplified template is shown as SEQ ID NO: 11.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 56℃for 90s, extension at 72℃for 30s,30 cycles; extending at 72deg.C for 7min, and storing at 15deg.C.
The PCR reaction system is as follows: dH (dH) 2 O(14.2μL)，10×KOD Buffer(2μL)，KOD(0.2μL)，dNTP(1.6μL)，cDNA(1μL，1.0μg/μL)，F-BamH I(0.5μL)，R-Hind III(0.5μL)。
The PCR product is detected by 1.5% agarose gel electrophoresis, the target band is recovered by cutting gel, and the P1h3 fragment is obtained by clone sequencing.
(2) Construction of recombinant expression vector pcDNA3.1-P1h3-Lamp2b
And simultaneously, carrying out BamH I and HindIII double enzyme digestion on the P1h3 fragment and the recombinant plasmid pcDNA3.1-Lamp2b containing the Lamp2b gene (purchased source: nanjing Jinsrey technology company), recovering enzyme digestion products, and connecting the two fragments by Taq DNA ligase to obtain the constructed recombinant expression vector pcDNA3.1-P1h3-Lamp2b, wherein the connecting system is 4 mu L of pcDNA3.1-Lamp2b fragment, 1 mu L of P1h3 fragment and 5 mu L of DNA ligase.
The reaction was allowed to react overnight at 16℃and then the ligation products were transformed into DH 5. Alpha. Competent cells overnight at 37℃and colonies were picked and verified by PCR. The positive clone strain is selected, and BamH I and HindIII are subjected to double enzyme digestion and identification, so that the expected recombinant expression vector pcDNA3.1-P1h3-Lamp2b is obtained.
S2, constructing a recombinant expression vector pcDNA3.1-GSDMD-N: the recombinant expression vector is completed by Nanjing Jinsri biotechnology company;
(1) Obtaining GSDMD-N Gene sequence
Designing a primer pair according to the GSDMD-N gene sequence, and amplifying the obtained product by polymerase chain reaction to obtain GSDMD-N, wherein the upstream primer is added with BamH I enzyme cutting sites and then has the sequence of F-BamH I:5'-GTGGGATCCGCCACCATGGGGTCGGCCTTTGAGCGGGT-3', as set forth in SEQ ID NO:3 is shown in the figure; the sequence after the downstream primer is added with HindIII cleavage site is R-HindIII: 5'-CACAAGCTTTCACTTGTCGTCATCGTCTTTGTAGTCAGGAAGTTGTGGAGG C-3', as set forth in SEQ ID NO: 4.
Wherein, the sequence of the amplified template is shown as SEQ ID NO: shown at 12.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 56℃for 90s, extension at 72℃for 30s,45 cycles; extending at 72deg.C for 10min, and preserving at 15deg.C.
The PCR reaction system is as follows: dH (dH) 2 O(14.2μL)，10×KOD Buffer(2μL)，KOD(0.2μL)，dNTP(1.6μL)，cDNA(1μL，1.0μg/μL)，F-BamH I(0.5μL)，R-Hind III(0.5μL)。
And detecting the PCR product by 1.5% agarose gel electrophoresis, cutting the gel to recover a target band, and cloning and sequencing to obtain the GSDMD-N fragment.
(2) Construction of recombinant expression vector pcDNA3.1-GSDMD-N
And simultaneously, carrying out BamH I and HindIII double enzyme digestion on the GSDMD-N fragment and the recombinant plasmid vector pcDNA3.1, recovering enzyme digestion products, and connecting the two fragments by Taq DNA ligase to obtain the constructed recombinant expression vector pcDNA3.1-GSDMD-N, wherein the connecting system is 4 mu L of the pcDNA3.1 fragment, 1 mu L of the GSDMD-N fragment and 5 mu L of DNA ligase.
The reaction was allowed to react overnight at 16℃and then the ligation products were transformed into DH 5. Alpha. Competent cells overnight at 37℃and colonies were picked and verified by PCR. And selecting a positive clone strain, and carrying out double enzyme digestion identification on BamH I and HindIII to obtain a recombinant expression vector pcDNA3.1-GSDMD-N.
S3, screening of puromycin concentration
(1) After 0.5. Mu.g of the recombinant expression vector pcDNA3.1-GSDMD-N was mixed with 1. Mu.L of the transfection reagent, the mixture was allowed to stand at 37℃for 20 minutes, and then a 96-well plate without double antibody medium (DMEM, gibco, U.S. A.; fetal bovine serum, gibco, U.S. A.) was slowly added thereto, and 0. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 6. Mu.g/mL, 8. Mu.g/mL, 10. Mu.g/mL puromycin was added thereto, and 6 wells were set for each concentration;
(2) The supernatant was discarded, CCK8 reagent was added, OD was measured at 450nm, statistical difference analysis was performed using GraphPad software, and the optimal puromycin concentration was selected.
As shown in FIG. 1, the optimal puromycin concentration for inhibiting translation of the recombinant expression vector pcDNA3.1-GSDMD-N is 4 μg/mL.
S4、EV GSDMD-N Is prepared from
(1) The cell is transfected by a recombinant expression vector of a tumor targeting protein HER2 single-chain antibody (namely, the recombinant expression vector pcDNA3.1-P1h3-Lamp2 b), and the specific operation process is as follows:
2-4 mug recombinant expression vector pcDNA3.1-P1h3-Lamp2b is mixed with 4-8 mug transfection reagent, and then is slowly added into a 293T culture dish without double antibody culture medium (DMEM, gibco company, USA; fetal bovine serum, gibco company, USA) after being kept stand at 37 ℃ for 20 min;
(2) The method comprises the following steps of transfecting a recombinant expression vector (namely the recombinant expression vector pcDNA3.1-GSDMD-N) containing a cell death related effector molecule GSDMD-N plasmid into cells of a recombinant expression vector (namely the recombinant expression vector pcDNA3.1-P1h3-Lamp2 b) transfected with a tumor targeting protein HER2 single-chain antibody:
after 24h, 2-4. Mu.g of recombinant expression vector pcDNA3.1-GSDMD-N was mixed with 4-8. Mu.L of transfection reagent, and after standing at 37℃for 20min, the mixture was slowly added to a cell culture dish containing no double antibody medium (DMEM, gibco, USA; fetal bovine serum, gibco, USA) and 4. Mu.g/mL puromycin was added to the cell culture dish containing transfected recombinant expression vector pcDNA3.1-P1h3-Lamp2 b;
(3) Placing at 37deg.C with 5% CO 2 After 6h, the cells were replaced with serum-free complete medium (DMEM, gibco, USA) and after 48h the cell supernatants were collected;
(4) Filtering the cell supernatant with a 0.22 μm filter to remove cell debris;
(5) The cell supernatant is subjected to ultracentrifugation for 10 hours at 100000 Xg, the obtained precipitate is EVs containing more effector molecule mRNA, and the EVs and Ce6 are incubated for 12 hours according to the mass ratio of 1:10, thus obtaining the EV GSDMD-N 。
Example 2
Acquisition of extracellular vesicle co-delivery System targeting EGFR-positive tumor cells with the apoptosis-related effector molecule GSDME-N mRNA
The specific operation process is as follows:
s1, constructing a recombinant expression vector pcDNA3.1-GE11: the recombinant expression vector is completed by Nanjing Jinsri biotechnology company;
(1) Obtaining the GE11 Gene sequence
Designing a primer pair based on the original unannotated sequence of the existing GE11 gene (Genbank accession: NC_ 000007.14), and taking the GE11 genome as a template, amplifying the obtained product by polymerase chain reaction to obtain GE11, wherein the sequence of the upstream primer after adding BamHI enzyme cutting sites is F-BamHI: 5'-GTGGGATCCGCCACCCTGGAGTCGACTTATGAGGGGAT-3', as set forth in SEQ ID NO:5 is shown in the figure; the sequence after the downstream primer is added with HindIII cleavage site is R-HindIII: 5'-CACAAGCTTACACTCGTCGCCATCCTCTATGTAGTCTGGAGGC-3', as set forth in SEQ ID NO: shown at 6.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 56℃for 90s, extension at 72℃for 30s,30 cycles; extending at 72deg.C for 8min, and storing at 15deg.C.
The PCR reaction system is as follows: dH (dH) 2 O(14.2μL)，10×KOD Buffer(2μL)，KOD(0.2μL)，dNTP(1.6μL)，cDNA(1μL,1.0μg/μL)，F-BamH I(0.5μL)，R-Hind III(0.5μL)。
Detecting the PCR product by 1.5% agarose gel electrophoresis, cutting the gel to recover a target band, and obtaining the GE11 fragment by clone sequencing.
(2) Construction of recombinant expression vector pcDNA3.1-GE11
Meanwhile, bamH I and Hind III double enzyme digestion are carried out on the GE11 fragment and the recombinant plasmid pcDNA3.1-Lamp2b containing the Lamp2b gene (purchased source: nanjing Jinsri technology company), enzyme digestion products are recovered by gel digestion, taq DNA ligase is connected with the two fragments, thus obtaining the constructed recombinant expression vector pcDNA3.1-GE11, the connection system is pcDNA3.1-Lamp2b 4 mu L, GE11 fragment 1 mu L and DNA ligase 5 mu L.
The reaction was allowed to react overnight at 16℃and then the ligation products were transformed into DH 5. Alpha. Competent cells overnight at 37℃and colonies were picked and verified by PCR. The positive clone strain is selected, and BamH I and HindIII are subjected to double enzyme digestion and identification, so that a recombinant expression vector pcDNA3.1-GE11 is obtained.
S2, constructing a recombinant expression vector pcDNA3.1-GSDME-N: the recombinant expression vector is completed by Nanjing Jinsri biotechnology company;
(1) Obtaining GSDME-N Gene sequence
Designing a primer pair, and amplifying the obtained product by polymerase chain reaction to obtain GSDME-N, wherein the sequence of the upstream primer added with BamH I enzyme cutting site is F-BamH I:5'-GCGGGATCCCGCCACCATGTTTGCCAAAGCAACCAGGAA-3', as set forth in SEQ ID NO: shown in figure 7; the sequence after the downstream primer is added with HindIII cleavage site is R-HindIII: 5'-CGCAAGCTTTCACTTGTCGTCATCGTCTTTGTAGTCATCTGGCATGTCTATGA ATGCA-3', as set forth in SEQ ID NO: shown at 8.
Wherein, the sequence of the amplified template is shown as SEQ ID NO: shown at 13.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 56℃for 90s, extension at 72℃for 30s,30 cycles; extending at 72deg.C for 3min, and preserving at 15deg.C.
The PCR reaction system is as follows: dH (dH) 2 O(14.2μL)，10×KOD Buffer(2μL)，KOD(0.2μL)，dNTP(1.6μL)，cDNA(1μL,1.0μg/μL)，F-BamH I(0.5μL)，R-Hind III(0.5μL)。
Detecting the PCR product by 1.5% agarose gel electrophoresis, cutting the gel to recover a target band, and obtaining the GSDME-N fragment by clone sequencing.
(2) Construction of recombinant expression vector pcDNA3.1-GSDME-N
And simultaneously, carrying out BamH I and HindIII double enzyme digestion on the GSDME-N fragment and the recombinant plasmid vector pcDNA3.1, recovering enzyme digestion products, and connecting the two fragments by Taq DNA ligase to obtain the constructed recombinant expression vector pcDNA3.1-GSDME-N, wherein the connecting system is 4 mu L of the pcDNA3.1 fragment, 1 mu L of the GSDMD-N fragment and 5 mu L of DNA ligase.
The reaction was allowed to react overnight at 16℃and then the ligation products were transformed into DH 5. Alpha. Competent cells overnight at 37℃and colonies were picked and verified by PCR. And selecting positive clone strains, and carrying out double enzyme digestion identification on BamH I and HindIII to obtain a recombinant expression vector pcDNA3.1-GSDME-N.
S3, screening of puromycin concentration
(1) After 0.5. Mu.g of the recombinant expression vector pcDNA3.1-GSDME-N was mixed with 1. Mu.L of the transfection reagent, the mixture was allowed to stand at 37℃for 20 minutes, and then a 96-well plate without double antibody medium (DMEM, gibco, U.S. A.; fetal bovine serum, gibco, U.S. A.) was slowly added thereto, and 0. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 6. Mu.g/mL, 8. Mu.g/mL, 10. Mu.g/mL puromycin was added thereto, and 6 wells were set for each concentration;
(2) The supernatant was discarded, CCK8 reagent was added, OD was measured at 450nm, statistical difference analysis was performed using GraphPad software, and the optimal puromycin concentration was selected.
The optimal puromycin inhibition concentration obtained by screening is 4 mug/mL.
S4、EV GSDME-N Is prepared from
(1) The cell is transfected by a recombinant expression vector of tumor targeting peptide GE11 peptide (namely, the recombinant expression vector pcDNA3.1-GE 11) and the specific operation process is as follows:
mixing 2-4 mu recombinant expression vector pcDNA3.1-GE11 with 4-8 mu L transfection reagent, standing at 37 ℃ for 20min, and slowly adding into 293T culture dish without double antibody culture medium (DMEM, gibco company, U.S. A.; fetal bovine serum, gibco company, U.S. A.);
(2) The method comprises the following steps of transfecting a recombinant expression vector (namely, the recombinant expression vector pcDNA3.1-GSDME-N) containing a cell death related effector molecule GSDME-N plasmid into cells of the recombinant expression vector (namely, the recombinant expression vector pcDNA3.1-GE 11) transfected with tumor targeting peptide GE11 peptide:
after 24h, 2-4. Mu.g of recombinant expression vector pcDNA3.1-GSDME-N was mixed with 4-8. Mu.L of transfection reagent, and after standing at 37℃for 20min, the mixture was slowly added to a cell culture dish containing transfected recombinant expression vector pcDNA3.1-GE11 without double antibody medium (DMEM, gibco, USA; fetal bovine serum, gibco, USA) and 4. Mu.g/mL puromycin was added;
(3) Placing at 37deg.C with 5% CO 2 After 4-6 hours, changing to serum-free complete culture medium (DMEM, gibco company, USA), and collecting cell supernatant after 48 hours;
(4) Filtering the cell supernatant with a 0.22 μm filter to remove cell debris;
(5) The cell supernatant is subjected to ultracentrifugation for 10 hours at 100000 Xg, the obtained precipitate is EVs containing more effector molecule mRNA, and the EVs and Ce6 are incubated for 12 hours according to the mass ratio of 1:10, thus obtaining the EV GSDME-N 。
Example 3
Acquisition of extracellular vesicle co-delivery System targeting HER 2-positive tumors with apoptosis-related effector molecule tBId mRNA
The specific operation process is as follows:
s1, constructing a recombinant expression vector pcDNA3.1-P1h3-Lamp2b: the recombinant expression vector is completed by Nanjing Jinsri biotechnology company; the construction process of the recombinant expression vector pcDNA3.1-P1h3-Lamp2b in example 1 is the same;
s2, constructing a recombinant expression vector pcDNA3.1-tBID: the recombinant expression vector is completed by Nanjing Jinsri biotechnology company;
(1) Obtaining tBId Gene sequence
Designing a primer pair according to a tBID gene sequence, and amplifying the obtained product by polymerase chain reaction to obtain tBID, wherein the upstream primer is added with a BamHI enzyme cutting site and has the sequence of F-BamHI: 5'-GTGGGATCCCGCCACCATGTTCAGCGTATTTGAGGAAAT-3', as set forth in SEQ ID NO: shown as 9; the sequence after the downstream primer is added with HindIII cleavage site is R-HindIII: 5'-GTGAAGCTTTCACTTGTCGTCATCGTCTTTGTAGTCCTCCATGTCCTCCAAC TTCTCCT-3', as set forth in SEQ ID NO: shown at 10.
Wherein, the sequence of the amplified template is shown as SEQ ID NO: 14.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 56℃for 90s, extension at 72℃for 30s,30 cycles; extending at 72deg.C for 8min, and storing at 15deg.C.
The PCR reaction system is as follows: dH (dH) 2 O(14.2μL)，10×KOD Buffer(2μL)，KOD(0.2μL)，dNTP(1.6μL)，cDNA(1μL，1.0μg/μL)，F-BamH I(0.5μL)，R-Hind III(0.5μL)。
The PCR product is detected by 1.5% agarose gel electrophoresis, the target band is recovered by cutting gel, and the tBId fragment is obtained by cloning and sequencing.
(2) Construction of recombinant expression vector pcDNA3.1-tBId
And simultaneously carrying out BamH I and Hind III double enzyme digestion on the tBID fragment and the recombinant plasmid vector pcDNA3.1, recovering enzyme digestion products, and connecting the two fragments by Taq DNA ligase to obtain the constructed recombinant expression vector pcDNA3.1-tBID, wherein the connecting system is 4 mu L of the pcDNA3.1 fragment, 1 mu L of the tBID fragment and 5 mu L of DNA ligase.
The reaction was allowed to react overnight at 16℃and then the ligation products were transformed into DH 5. Alpha. Competent cells overnight at 37℃and colonies were picked and verified by PCR. And (3) selecting a positive clone, and carrying out double enzyme digestion identification on BamH I and HindIII to obtain a recombinant expression vector pcDNA3.1-tBId.
S3, screening of puromycin concentration
(1) After 0.5. Mu.g of the recombinant expression vector pcDNA3.1-tBId was mixed with 1. Mu.L of the transfection reagent, the mixture was allowed to stand at 37℃for 20 minutes, and then a 96-well plate containing no double antibody medium (DMEM, gibco, U.S.A.; fetal bovine serum, gibco, U.S.A.) was slowly added thereto, and 0. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 6. Mu.g/mL, 8. Mu.g/mL, 10. Mu.g/mL puromycin was added thereto, each concentration being set at 6 wells;
(3) The supernatant was discarded, CCK8 reagent was added, OD was measured at 450nm, statistical difference analysis was performed using GraphPad software, and the optimal puromycin concentration was selected.
The optimal puromycin inhibition concentration obtained by screening is 5 mug/mL.
S4、EV tBid Is prepared from
(1) The cell is transfected by a recombinant expression vector of a tumor targeting protein HER2 single-chain antibody (namely, the recombinant expression vector pcDNA3.1-P1h3-Lamp2 b), and the specific operation process is as follows:
2-4 mug recombinant expression vector pcDNA3.1-P1h3-Lamp2b is mixed with 4-8 mug transfection reagent, and then is slowly added into a 293T culture dish without double antibody culture medium (DMEM, gibco company, USA; fetal bovine serum, gibco company, USA) after being kept stand at 37 ℃ for 20 min;
(2) The specific operation process of transfecting the recombinant expression vector (namely the recombinant expression vector pcDNA3.1-tBId) containing the cell death related effector molecule tBId plasmid into cells of the recombinant expression vector (namely the recombinant expression vector pcDNA3.1-P1h3-Lamp2 b) transfected with the tumor targeting protein HER2 single-chain antibody is as follows:
after 24h, 2-4. Mu.g of recombinant expression vector pcDNA3.1-tBId is mixed with 4-8. Mu.L of transfection reagent, and after standing for 20min at 37 ℃, the mixture is slowly added into a cell culture dish without double-antibody culture medium (DMEM, gibco company, USA; fetal bovine serum, gibco company, USA), and 5. Mu.g/mL of puromycin concentration is added;
(3) Placing at 37deg.C with 5% CO 2 After 4-6 hours, changing to serum-free complete culture medium (DMEM, gibco company, USA), and collecting cell supernatant after 48 hours;
(4) Filtering the cell supernatant with a 0.22 μm filter to remove cell debris;
(5) The cell supernatant is subjected to ultracentrifugation for 10 hours at 100000 Xg, the obtained precipitate is EVs containing more effector molecule mRNA, and the EVs and Ce6 are incubated for 12 hours according to the mass ratio of 1:10, thus obtaining the EV tBid 。
Example 4
Acquisition of extracellular vesicle co-delivery System targeting HER 2-positive tumors with the apoptosis-related effector molecule GSDMD-N mRNA
The specific operation process is as follows:
s1, constructing a recombinant expression vector pcDNA3.1-P1h3-Lamp2b: the recombinant expression vector is completed by Nanjing Jinsri biotechnology company;
(1) Obtaining the P1h3 Gene sequence
Designing a primer pair, and amplifying the obtained product by polymerase chain reaction to obtain P1h3, wherein the sequence of the upstream primer added with BamH I enzyme cutting site is F-BamH I:5'-CTCGGATCCGCGCCACCATGACCTGCATGTTGGAACGCAT-3', as set forth in SEQ ID NO:1 is shown in the specification; the sequence after the downstream primer is added with HindIII cleavage site is R-HindIII: 5' -CTCAA GCTTGGCTCACTTGTCGTCATCGTCTTTGTAGTCCCCCTCACTCCT CGCAGCACAGT-3', as set forth in SEQ ID NO: 2.
Wherein, the sequence of the amplified template is shown as SEQ ID NO: 11.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 56℃for 90s, extension at 72℃for 30s,30 cycles; extending at 72deg.C for 7min, and storing at 15deg.C.
The PCR reaction system is as follows: dH (dH) 2 O(14.2μL)，10×KOD Buffer(2μL)，KOD(0.2μL)，dNTP(1.6μL)，cDNA(1μL)，F-BamH I(0.5μL)，R-Hind III(0.5μL)。
The PCR product is detected by 1.5% agarose gel electrophoresis, the target band is recovered by cutting gel, and the P1h3 fragment is obtained by clone sequencing.
(2) Construction of recombinant expression vector pcDNA3.1-P1h3-Lamp2b
And simultaneously, carrying out BamH I and Hind III double enzyme digestion on the P1h3 fragment and the recombinant plasmid pcDNA3.1-Lamp2b containing the Lamp2b gene, recovering enzyme digestion products, and connecting the two fragments by Taq DNA ligase to obtain the constructed recombinant expression vector pcDNA3.1-P1h3-Lamp2b, wherein the connecting system is 4 mu L of the pcDNA3.1-Lamp2b fragment, 1 mu L of the P1h3 fragment and 5 mu L of the DNA ligase.
The reaction was allowed to react overnight at 16℃and then the ligation products were transformed into DH 5. Alpha. Competent cells overnight at 37℃and colonies were picked and verified by PCR. The positive clone strain is selected, and BamH I and HindIII are subjected to double enzyme digestion and identification, so that the expected recombinant expression vector pcDNA3.1-P1h3-Lamp2b is obtained.
S2, constructing a recombinant expression vector pcDNA3.1-GSDMD-N: the recombinant expression vector is completed by Nanjing Jinsri biotechnology company;
(1) Obtaining GSDMD-N Gene sequence
Designing a primer pair according to the GSDMD-N gene sequence, and amplifying the obtained product by polymerase chain reaction to obtain GSDMD-N, wherein the upstream primer is added with BamH I enzyme cutting sites and then has the sequence of F-BamH I:5'-GTGGGATCCGCCACCATGGGGTCGGCCTTTGAGCGGGT-3', as set forth in SEQ id no:3 is shown in the figure; the sequence after the downstream primer is added with HindIII cleavage site is R-HindIII: 5'-CACAAGCTTTCACTTGTCGTCATCGTCTTTGTAGTCAGGAAGTTGTGGAGG C-3', as set forth in SEQ ID NO: 4.
Wherein, the sequence of the amplified template is shown as SEQ ID NO: shown at 12.
The PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 56℃for 90s, extension at 72℃for 30s,45 cycles; extending at 72deg.C for 10min, and preserving at 15deg.C.
The PCR reaction system is as follows: dH (dH) 2 O(14.2μL)，10×KOD Buffer(2μL)，KOD(0.2μL)，dNTP(1.6μL)，cDNA(1μL)，F-BamH I(0.5μL)，R-Hind III(0.5μL)。
(2) Construction of recombinant expression vector pcDNA3.1-GSDMD-N
And simultaneously, carrying out BamH I and HindIII double enzyme digestion on the GSDMD-N fragment and the recombinant plasmid vector pcDNA3.1, recovering enzyme digestion products, and connecting the two fragments by Taq DNA ligase to obtain the constructed recombinant expression vector pcDNA3.1-GSDMD-N, wherein the connecting system is 4 mu L of the pcDNA3.1 fragment, 1 mu L of the GSDMD-N fragment and 5 mu L of DNA ligase.
The reaction was allowed to react overnight at 16℃and then the ligation products were transformed into DH 5. Alpha. Competent cells overnight at 37℃and colonies were picked and verified by PCR. And selecting a positive clone strain, and carrying out double enzyme digestion identification on BamH I and HindIII to obtain a recombinant expression vector pcDNA3.1-GSDMD-N.
S3, screening of puromycin concentration
(1) After 0.5. Mu.g of the recombinant expression vector pcDNA3.1-GSDMD-N was mixed with 1. Mu.L of the transfection reagent, the mixture was allowed to stand at 37℃for 20 minutes, and then a 96-well plate without double antibody medium (DMEM, gibco, U.S. A.; fetal bovine serum, gibco, U.S. A.) was slowly added thereto, and 0. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL, 6. Mu.g/mL, 8. Mu.g/mL, 10. Mu.g/mL puromycin was added thereto, and 6 wells were set for each concentration;
(2) The supernatant was discarded, CCK8 reagent was added, OD was measured at 450nm, statistical difference analysis was performed using GraphPad software, and the optimal puromycin concentration was selected.
S4、EV GSDMD-N Is prepared from
(1) The cell is transfected by a recombinant expression vector of a tumor targeting protein HER2 single-chain antibody (namely, the recombinant expression vector pcDNA3.1-P1h3-Lamp2 b), and the specific operation process is as follows:
2-4 mug recombinant expression vector pcDNA3.1-P1h3-Lamp2b is mixed with 4-8 mug transfection reagent, and then is slowly added into a 293T culture dish without double antibody culture medium (DMEM, gibco company, USA; fetal bovine serum, gibco company, USA) after being kept stand at 37 ℃ for 20 min;
(2) The method comprises the following steps of transfecting a recombinant expression vector (namely the recombinant expression vector pcDNA3.1-GSDMD-N) containing a cell death related effector molecule GSDMD-N plasmid into cells of a recombinant expression vector (namely the recombinant expression vector pcDNA3.1-P1h3-Lamp2 b) transfected with a tumor targeting protein HER2 single-chain antibody:
after 24h, 2-4. Mu.g of recombinant expression vector pcDNA3.1-GSDMD-N was mixed with 4-8. Mu.L of transfection reagent, and after standing at 37℃for 40min, the mixture was slowly added to a cell culture dish containing no double antibody medium (DMEM, gibco, USA; fetal bovine serum, gibco, USA) and 4. Mu.g/mL puromycin was added to the cell culture dish containing transfected recombinant expression vector pcDNA3.1-P1h3-Lamp2 b;
(3) Placing at 37deg.C with 5% CO 2 After 4h, the cell incubator was replaced with serum-free complete medium (DMEM, gibco, USA),collecting cell supernatant after 48 hours;
(4) Filtering the cell supernatant with a 0.22 μm filter to remove cell debris;
(5) The cell supernatant is subjected to ultracentrifugation for 16h at 100000 Xg, the obtained precipitate is EVs containing more effector molecule mRNA, and the EVs and purpin 18 are incubated for 24h according to the mass ratio of 1:50, thus obtaining the EV GSDMD-N 。
The extracellular vesicle co-delivery systems prepared in examples 1 to 4 described above are substantially similar in terms of tumor inhibition effect, so that the extracellular vesicle co-delivery system EV prepared in example 1 alone is described below GSDMD-N The effect will be described for the example.
According to EV in example 1 GSDMD-N The preparation method of (2) synchronously collecting normal 293T-derived extracellular vesicles, namely, exchanging a 293T culture dish with a serum-free complete medium (DMEM, gibco company, USA), and collecting cell supernatants after 48 hours; filtering the cell supernatant with a 0.22 μm filter to remove cell debris; the cell supernatant was ultracentrifuged for 10h at 100000 Xg to obtain a pellet as a control extracellular vesicle.
And performing Westernblot detection on the extracted extracellular vesicles to obtain P1h3 expression conditions, and comparing the control extracellular vesicles.
According to the method for extracting extracellular vesicles in example 1, extracellular vesicles EV containing more effector mRNA were collected GSDMD-N Removing residual liquid by vacuum pump, adding 50 μl RIPA lysate (Biyun, china), blowing with a pipette until extracellular vesicles are completely dissolved, and standing on ice for 30min to allow extracellular vesicle proteins to be completely lysed. Protein quantification (Thermo, usa) and protein sample preparation were performed by the double dilution method. The gel (Biyun, china) with 12% content is prepared by self, and then the extracellular vesicle protein sample is added for electrophoresis. Setting electrophoresis voltage 90V for 30min, and starting electrophoresis. And after the protein sample enters the lower separation gel, adjusting the electrophoresis voltage to 120V for 90min. And observing the position of the protein sample, stopping electrophoresis after reaching the expected position, turning to transfer the membrane, and setting the current to be 200mA for 120min. Followed by antibody incubation. Diluting proper amount of antibody according to the specification of the antibody, and selecting primary antibodyThe Lamp2b antibody (Abcam, usa) was selected, slowly shaken on a shaker for 30min, and then placed in a refrigerator at 4 ℃ for overnight incubation. Goat anti-rabbit IgG HRP (BBI Producer, china) was selected as the secondary antibody, placed in a cassette, and slowly shaken on a shaker at room temperature for 1h. Finally, luminescence (ECL luminescence, GE, usa) was performed with GAPDH as an internal reference.
As shown in FIG. 2, 293T-derived extracellular vesicles carry the P1h3 motif.
The extracellular vesicle co-delivery system EV GSDMD-N Application of inhibiting tumor growth in SKBR3 tumor-bearing transplanted mouse model
Intratumoral injection treatment was performed by randomly dividing SKBR3 tumor-bearing transplanted mice model (ex tieka) into 3 groups, in PBS group (PBS purchased from Yu Kaiji organism, KGB5001, control), control extracellular vesicle group (control extracellular vesicles extracted in example 1), EV GSDMD-N Group (modified extracellular vesicles EV extracted in example 1) GSDMD-N )。
PBS group: PBS was injected into SKBR3 tumor bearing transplanted mice at a dose of 100 μg/200 μl each, 1 time every two days for four times, followed by tumor taking observation;
control extracellular vesicle set (designated EV Ctrl ): control extracellular vesicles were injected into SKBR3 tumor bearing transplanted mice at a dose of 100 μg/200 μl each, 1 time every two days, four times total, followed by tumor taking observations;
EV GSDMD-N group (denoted EV) Tx ): EV was dosed at 100. Mu.g/200. Mu.L each GSDMD-N Injection into SKBR3 tumor-bearing transplanted mice was performed with ultrasound 1 every two days four times, followed by tumor taking observations.
The results are shown in FIG. 3, EV GSDMD-N Compared with PBS group and control extracellular vesicle group, the SKBR3 tumor-bearing transplanted mice have obviously reduced tumor, which indicates that the extracellular vesicle co-delivery system provided by the invention can effectively inhibit the tumor growth.
While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.
Claims (10)
1. An extracellular vesicle co-delivery system for tumor treatment, comprising:
s1, transfecting cells with a recombinant expression vector of a tumor targeting protein or targeting peptide to obtain cells transfected with the recombinant expression vector of the tumor targeting protein or targeting peptide;
s2, transfecting a recombinant expression vector containing a cell death related effector molecule plasmid into cells transfected with tumor targeting proteins or targeting peptides in S1, adding purinin to inhibit translation of the transfected effector molecule plasmid, and centrifuging to obtain extracellular vesicles with surface modified effector molecule mRNA;
and S3, incubating the extracellular vesicles obtained in the step S2 with a substance capable of generating active oxygen to obtain an extracellular vesicle co-delivery system.
2. The extracellular vesicle co-delivery system of claim 1, wherein the tumor targeting protein or targeting peptide is a HER2 single chain antibody or a GE11 peptide.
The effector molecule related to cell death is one of tbid, GSDMD-N, GSDME-N or GSDMB-N.
3. The extracellular vesicle co-delivery system according to claim 2, wherein,
the recombinant expression vector of the tumor targeting protein or targeting peptide is obtained by loading the tumor targeting protein or targeting peptide between BamHI and HindIII cleavage sites of a recombinant plasmid pcDNA3.1-Lamp2b containing Lamp2b genes;
the recombinant expression vector containing the cell death related effector molecule plasmid is obtained by loading the effector molecule plasmid between BamHI and HindIII cleavage sites of a pcDNA3.1 (+) vector.
4. The extracellular vesicle co-delivery system according to claim 3, wherein,
the transfection in S1 is to mix the recombinant expression vector of the tumor targeting protein or targeting peptide with the transfection reagent according to the ratio of 1-2 mug to 2-4 mug, and then to add the mixture into a cell culture system without double antibody culture medium for culturing for 24h after standing for 20-40 min at 37 ℃;
the transfection in S2 is to mix the recombinant expression vector containing the cell death related effector molecule plasmid with a transfection reagent according to the ratio of 1-2 mug to 2-4 mug, and then to add the mixture into a cell culture system of the recombinant expression vector transfected with tumor targeting protein or targeting peptide in S1 after standing for 20-40 min at 37 ℃.
5. The extracellular vesicle co-delivery system according to claim 4, wherein,
the specific operation process for inhibiting the translation of the transfected effector molecule plasmid is as follows:
after adding the purine, incubating for 4-6 hours at 37 ℃, and continuously incubating for 48 hours in a complete medium without serum, and collecting cell supernatant;
the centrifugation is to use 100000 Xg ultracentrifugation for 10-16 h on cell supernatant from which cell debris is removed.
6. The extracellular vesicle co-delivery system according to claim 5, wherein said purlin has an inhibitory concentration of 4 μg/mL.
7. The extracellular vesicle co-delivery system according to claim 1, wherein the substance capable of generating active oxygen is a sonosensitizer or a photosensitizer.
8. The extracellular vesicle co-delivery system of claim 7, wherein the sonosensitizer is chlorin E6 or purpin 18.
9. The extracellular vesicle co-delivery system according to claim 1, wherein the co-incubation is performed by mixing the extracellular vesicle obtained by S2 with a substance capable of generating active oxygen in a mass ratio of 1:10-50 for 12-24 hours.
10. Use of the extracellular vesicle co-delivery system of any one of claims 1-9 in the preparation of an anti-tumor drug.
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